This invention relates to a new leukocyte specific and immunoregulatory protein (LST-1) and to the corresponding protein produced by recombinant techniques as well as nucleic acids which code for proteins with LST-1 activity, methods of use and of production.
Cytokines are proteins with a molecular weight of less than 50 kD, which mediate the exchange of autocrine, paracrine or endocrine signals between the cellular components of tissues or between different tissues. The cytokines identified so far include growth factors, interleukins and interferons and act on cells in many systems of the body: the hematopoietic system, the immune system, the nervous system, the skeletal system, connective tissues, and probably most other tissues and organs of the body (for reference see A. Thompson ed. (1991), The Cytokine Handbook, London, Academic Press and A. Miyajima et al., Annual Reviews Immunol. 10 (1992) 295-331). Examples of cytokines are EGF, NGF, PDGF, FGF, IL-1 to IL-7, GM-CSF, G-CSF, MCSF, IFN, TNF-xcex1, TG-xcex1 and -xcex2.
More than 100 cytokines have been identified so far, but there is a need for further new cytokines which might be important for potential health care advances.
Accordingly it is an object of the present invention to provide a new cytokine showing new biological properties.
It is a further object of the present invention to provide the new protein using recombinant DNA molecules capable of expressing said protein in order that the binding protein will be more readily available. These and other objects of the invention have been accomplished by providing a purified protein according to the invention, selected from a group consisting of a protein which is at least 85% homologous to the amino acid sequence SEQ ID NO:3 and fragments thereof, wherein said protein is capable of binding to an antibody specific for said protein or its cell surface receptor on leukocytes.
The invention comprises especially novel therapeutic compositions comprising recombinant proteins produced using nucleic acid sequences encoding proteins with LST-1 activity. The LST-1 cDNA which can be used for the production of the recombinant protein was initially isolated from U-937 cells (DSM ACC 5) stimulated with IFN-xcex3. RT-PCR cloning of mMRA from these cells resulted in a cDNA clone designated pLST-1 having a length of 636 bp. Analysis of the LST-1 cDNA reveals that LST-1 consists of six exons. These exons are designated SEQ ID NO:1 exons 1A (bp 48-162), 1B (bp 544-652), 2 (bp 1044-1162), 3 (bp 1475-1567), 4 (bp 1775-1797) and 5 (bp-2325-2709). In SEQ ID NO:2 exon 5, after position 2345, there is an internal 5xe2x80x2 donor splice site. By alternative splicing, thus, two isoforms of the LST-1 protein can be formed, which accordingly have a length of either 104 or 97 amino acids (cf. SEQ ID NOS: 3 and 4). The human pLST-1 cDNA done contains an extended 5xe2x80x2 region encoding stop codons.
The invention is based on a new cytokine-like protein (denoted LST-1 protein, or LST-1, in the following) whose production is stimulated in U-937 cells by IFN-xcex3 by a factor of more than 100 preferably 1000, which binds to the surface of leukocytes and which
a) is coded by the DNA sequence shown in SEQ ID NO:2 for the mature protein or by the genomic sequence shown in SEQ ID NO:1,
b) is coded by DNA sequences which hybridize under stringent conditions with the DNA sequences shown in SEQ ID NO:1 or 2 or fragments of the DNA sequences in the DNA region which codes for the mature protein, or
c) is coded by DNA sequences which, if there was no degeneracy of the genetic code, would hybridize with the sequences defined in a) or b) and code for a polypeptide with amino acid sequence,
d) and the reading frame of said protein is defined by ATG starting at position 1144 of SEQ ID NO:1 following within no shift of the reading frame in the protein coding region following said ATG.
The protein can be defined by its DNA sequence (preferably by its cDNA sequence deduced from SEQ ID NO:1) and by the amino acid sequence derived therefrom. The LST-1 protein can occur in natural allelic variations which differ from individual to individual. Such variations of the amino acids are usually amino acid substitutions. However, they may also be deletions, insertions or additions of amino acids to the total sequence. The LST-1 protein according to the inventionxe2x80x94depending, both in respect of the extent and type, on the cell and cell type in which it is expressedxe2x80x94can be in glycosylated or non-glycosylated form The LST-1 proteins according to SEQ ID NO:3 and SEQ ID NO:4 are preferred.
LST-1 mRNA is expressed constitutively in T cells, macrophages, U-937 and at low levels in human tonsilla, lung and liver, which may be due to the lymphocytes and macrophages present in these tissues the protein shows an immunoregulatory activity. Transcription can be strongly enhanced by IFN-xcex3 in U-937 cells. Upon stimulation of the Jurkat T cell line (ATCC TIB 152) with TPA (12-o-tetradecanoylphorbol-13-acetate) only a short induction of LST-1 mRNA was observed. An increase of mRNA expression starts after four hours with a peak after 8 hours of stimulation and further decrease after 24 hours of stimulation.
xe2x80x9cImmunoregulatory activityxe2x80x9d means that the protein modulates directly or non-directly the cooperation of T-cells with macrophages.
The binding to the surface of leucocytes is preferably estimated in vitro. Such methods are known in the state of the art.
The term xe2x80x9chybridize under stringent conditionsxe2x80x9d means that two nucleic acid fragments are capable of hybridization to one another under standard hybridization conditions described in Sambrook et al., xe2x80x9cExpression of cloned genes in E. colixe2x80x9d in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, 9.47-9.62 and 11.45-11.61.
More specifically, stringent conditions as used herein refer to hybridization in 1 mol/l NaCl 1% SDS and 10% dextransulfate. This is followed by two washes of the filter at room temperature of 5 minutes in 2xc3x97SSC and one final wash for 30 minutes. This final wash may be at 0.5xc3x97SSC, 0.1% SDS, more preferably at 0.2xc3x97SSC, 0.1% SDS and most preferably at 0.1xc3x97SSC, 0.1% SDS, final wash taking place at 65xc2x0 C. Those of ordinary skilled in the art will recognize to that other conditions will afford the same degrees of stringency and are encompassed by the phraseology xe2x80x9cunder stringent conditionsxe2x80x9d and are encompassed herein.
LST-1 is a protein which is active in its glycosylated or unglycosylated form. The unglycosylated form can be produced by recombinant technology in prokaryotic cells.
xe2x80x9cProteins with LST-1 activityxe2x80x9d means also proteins with minor amino acid variations but with substantially the same LST1 activity. Substantially the same means that the activities are of the same biological properties and preferably at least 85% homology in amino acid sequence. More preferably, the amino acid sequences are at least 90% identical. LST-1 can be purified from U-937 cells by affinity chromatography using a monoclonal antibody against LST-1. It is also preferred to use other known protein purification techniques, including immunoprecipitation, gel filtration, ion exchange, chromatography, chromatofocussing, isoelectric focussing, selective precipitation, electrophoresis, and the like. Fraction isolated during purification procedures can be analyzed for the presence of LST-1 activity by using LST-1 specific antibodies.
The protein according to the invention can also be produced by recombinant means. Non-glycosylated LST-1 protein is obtained when it is produced recombinantly in prokaryotes. With the aid of the nucleic acid sequences provided by the invention it is possible to search for the LST-1 gene or its variants in genomes of any desired cells (e.g. apart from human cells, also in cells of other mammals), to identify these and to isolate the desired gene coding for the LST-1 protein Such processes and suitable hybridization conditions are known to a person skilled in the art and are described, for example, by Sambrook, J., et al., xe2x80x9cExpression of cloned genes in E. colixe2x80x9d in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, and B. D. Hames, S. G. Higgins, Nucleic acid hybridisationxe2x80x94a practical approach (1985) IRL Press, Oxford, England. In this case the standard protocols described in these publications are usually used for the experiments.
The use of recombinant DNA technology enables the production of numerous LST-1 protein derivatives. Such derivatives can, for example, be modified in individual or several amino acids by substitution, deletion or addition. The derivatization can, for example, be carried out by means of site directed mtutagenesis. Such variations can be easily carried out by a person skilled in the art (J. Sambrook, B. D. Hames, loc. cit.). It merely has to be ensured that the characteristic properties of the LST-1 protein (inhibition of the aforementioned cell lines) are preserved. The invention therefore in addition concerns a LST-1 protein which is a product of a prokaryotic or eukaryotic expression of an exogenous DNA.
The invention concerns a nucleic acid molecule for use in securing expression in a prokaryotic or eukaryotic host cell of a LST-1 protein and is selected from the group comprising
a) DNA sequences shown in SEQ ID NO:1 and 2 or the complementary sequences,
b) nucleic acid sequences which hybridize under stringent conditions with one of the sequences from a),
c) nucleic acid sequences which, if there was no degeneracy of the genetic code, would hybridize with one of the sequences stated in a) or b),
d) and the reading frame of said protein is defined by ATG starting at position 1144 of SEQ ID NO:1 following within no shift of the reading frame in the protein coding region following said ATG.
With the aid of such nucleic acids coding for a LST-1 protein, the protein according to the invention can be obtained in a reproducible manner and in large amounts. For expression in prokaryotic or eukaryotic organisms, such as prokaryotic host cells or eukaryotic host cells, the nucleic acid is integrated into suitable expression vectors, according to methods familiar to a person skilled in the arm Such an expression vector preferably contains a regulatable/inducible promoter. These recombinant vectors are then introduced for the expression into suitable host cells such as, e.g., E. coli as a prokaryotic host cell or Saccharomyces cerevisiae, Terato carcinoma cell line PA-1 sc 9117 (Bxc3xcttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells, CHO or COS cells as eukaryotic host cells and the transformed or transduced host cells are cultured under conditions which allow an expression of the heterologous gene. The isolation of the protein can be carried out according to known methods from the host cell or from the culture supernatant of the host cell. Such methods are described for example by Ausubel I., Frederick M., Current Protocols in Mol. Biol. (1992), John Wiley and Sons, New York. Also in vitro reactivation of the protein may be necessary.
In addition the invention concerns a process for obtaining a LST-1 protein by isolation of the culture supernatant of the U-937 cell line by means of a gel chromatographic separation and purification of a fraction.
The detection of transformed or transduced host cells which recombinantly produce the LST-1 protein and the purification of the protein are preferably carried out by means of antibodies which bind to this protein. Such antibodies can be obtained in a simple manner according to known methods by using the protein according to the invention as an antigen or an immunogen.
The invention therefore in addition concerns the use of the protein with LST-1 activity according to the invention for the production of antibodies which bind to this protein.
Anti-LST-1 antibodies are produced by immunization on and appropriate vertebrate host with purified LST-1 or polypeptide derivatives of LST-1, preferably with an adjuvant. Said techniques are well-known in the literature and are described, for example, by Harlow and Lane eds., Antibodies: A laboratory manual (1988), Cold Spring Harbor Laboratories Press.
For this, animals which are usually used for this purpose, such as in particular, sheep, rabbits or mice, are immunized with the protein according to the invention and subsequently the antiserum is isolated from the immunized animals according to known methods or spleen cells of the immunized animals are fused with immortalized cells, such as e.g. myeloma cells, according to the method of Kxc3x6hler and Milstein (Nature 256 (1975) 495-497). Those cells which produce a monoclonal antibody at the LST-1 protein are selected from the hybridoma cells obtained in this way and cloned. The monoclonal or polyclonal antibodies obtained in this way can be bound to a support material, such as e.g. cellulose, for an imunoabsorptive purification of the melanomaxe2x80x94inhibiting protein Furthermore, antibodies of this kind can be used for the detection of the LST-1 protein in samples, such as e.g. cut tissue or body fluids.
The invention therefore additionally concerns antibodies against the LST-1 protein which are obtainable by immunizing an animal with a LST-1 protein and isolating the antibodies from the serum or spleen cells of the immunized animals.
It has in addition turned out that the LST-1 protein has an immunoregulatory activity.
The invention in addition concerns the use of a protein according to the invention for the production of a therapeutic agent which can be used in tumor therapy or as an immunoregulating agent
The protein according to the invention is processed, if desired together with the usually used auxiliary agents, fillers and/or additives, in a pharmaceutical formulation for the said therapeutic applications.
The invention therefore in addition concerns a therapeutic composition containing a LST-1 protein according to the invention and if desired together with the auxiliary agents, fillers and/or additives that are usually used.
The invention further concerns the use of sequences of the LST-1 gene, preferably nucleic acid molecules coding for a protein having LST-1 activity, or activating polynucleotides from the 5xe2x80x2 untranslated region, in gene therapy, and in particular, for the production of medicaments for gene therapy.
Gene therapy of somatic cells can be accomplished by using, e.g., retroviral vectors, other viral vectors, or by non-viral gene transfer (for clarity cf. T. Friedmann, Science 244 (1989) 1275; Morgan 1993, RAC DATA MANAGEMENT REPORT, June 1993).
Vector systems suitable for gene therapy are, for instance, retroviruses (Mulligan, R. C. (1991) in Nobel Symposium 8: Ethiology of human disease at the DNA level (Lindsten, J. and Pattersun Editors) 143-189, Raven Press), adeno associated virus (McLughlin, J. Virol. 62 (1988), 1963), vaccinia virus (Moss et al., Ann. Rev. Immunol. 5 (1987) 305), bovine papilloma virus (Rasmussen et al., Methods Enzymol. 139 (1987) 642) or viruses from the group of the herpes viruses such as Epstein Barr virus (Margolskee et al., Mol. Cell. Biol. 8 (1988) 2937) or herpes simplex virus.
There are also known non-viral delivery systems. For this, usually xe2x80x9cnudexe2x80x9d nucleic acid, preferably DNA, is used, or nucleic acid together with an auxilary such as, e.g., transfer reagents (liposomes, dendromers, polylysine-transferrine-conjugates (Felgner et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7413).
Another preferred method of gene therapy is based on homologous recombination. In this, either the gene coding for the LST-1 protein can be inserted in one or more copies into the genome of somatic cells and/or the LST-1 gene endogenously present in the cells can be modulated, preferably activated.
Methods of homologous recombination are described, e.g., in Kucherlapati, Proc. in Nucl. Acids Res. and Mol. Biol. 36 (1989) 301; Thomas et al., Cell 44 (1986) 419-428; Thomas and Capecchi, Cell 51 (1987) 503-512; Doetschman et al., Proc. Natd. Acad. Sci. USA 85 (1988) 8583-8587 and Doetschman et al., Nature 330 (1987) 576-578. In these methods, a portion of DNA to be integrated at a specific site in the genome (gene fragment of LST-1) is bound to a targeting DNA. The targeting DNA is a DNA which is complementary (homologous) to a region (preferably within or proximal to the LST-1 gene) of the genomic DNA When two homologous portions of a single-stranded DNA (e.g. the targeting DNA and the genomic DNA) are in close proximity to one another they will hybridize and form a double-stranded helix. Then the LST-1 gene fragment and the targeting DNA can be integrated into the genome by means of occurrence of recombination. This homologous recombination can be carried out both in vitro and in vivo (in the patient).
Preferably, there is used a DNA which codes for a protein having LST-1 activity, a fragment which inhibits LST-1 expression (knock-out sequence) or a fragment capable of activating, after integration of the genome of a cell, expression, in this cell, of a protein having LST-1 activity. Such a fragment may be, for example, a promoter and/or enhancer region which is heterologous to the corresponding LST-1 region or which, after integration into the LST-1 gene, activates the actually silent or to a little extent expressed LST-1 gene transcriptionally and/or translationally.
Thus, by means of this DNA, one or more LST-1 genes are newly introduced into the target cell, or the essentially transcriptionally silent gene in the genome of a mamma cell is activated in such fashion that the mammalian cell is enabled to produce endogenous LST-1 protein. To this end, a DNA construct is inserted into the genome by homologous recombination, the DNA construct comprising the following: a DNA regulatory element capable of stimulating expression of this gene if operatively linked thereto; and one or more DNA target segments which are homologous to a region in this genome, which region is within or proximal to this gene. This construct is inserted into the genome of the mammalian cell in such fashion that the regulatory segment is operatively linked to the gene which codes for the protein having LST-1 activity. Preferably, the construct further comprises amplifying sequences, especially if genes coding for proteins with LST-1 activity are inserted into the cell.
For the introduction of LST-1 genes into the target cells, the construct comprises a regulatory element, one or more LST-1 genes and one or more target segments. The target segments are chosen in such a way that they hybridize with an appropriate region of the genome, whereby, after homologous recombination, the inserted exogenous LST-1 genes are expressed.
There are known a large number of processes by which homologous recombination can be initiated. Preferably, homologous recombination takes place during DNA replication or mitosis of the cells. A DNA of this kind can be used for the production of an agent for therapeutic treatment of tumors or for the production of homologous or heterologous LST-1 protein in a host organism.
It is possible to provide a test on the basis of the nucleic acid sequences of the LST-1 protein provided by the invention which can be used to detect nucleic acids which code for LST-1 proteins. Such a test can for example be carried out in cells or cell lysates and by means of nucleic acid diagnostics. In this case the sample to be examined is brought into contact with a probe which would hybridize with the nucleic acid sequence coding for the LST-1 protein A hybridization between the probe and nucleic acids from the sample indicates the presence of expressed LST-1 proteins. Such methods are known to a person skilled in the art and are for example described in WO 89/06698, EP-A 0 200 362, U.S. Pat. No. 2,915,082, EP-A 0 063 879, EP-A 0 173 251, EP-A 0 128 018. In a preferred embodiment of the invention, the nucleic acid of the sample which codes for a LST-1 protein is amplified before testing, e.g. by the well-known PCR technique. A derivatized (labelled) nucleic acid probe is usually used in the field of nucleic acid diagnostics. This probe is brought into contact with a carrier-bound denatured DNA or RNA from the sample and in this process the temperature, ionic strength, pH value and other buffer conditions are selected in such a way thatxe2x80x94depending on the length of the nucleic acid sample and the resulting melting temperature of the expected hybridxe2x80x94the labelled DNA or RNA can bind to homologous DNA or RNA (hybridization, see also Southern, E. M., J. Mol. Biol. 98 (1975), 503-517; Wahl, G. M. et al., Proc. Natl. Acad. Sci. USA 76 (1979), 3683-3687). Suitable carriers are membranes or carriers materials based on nitrocellulose (e.g. Schleicher and Schxc3xcll, BA 85, Amersham Hybond, C.), reinforced or bound nitrocellulose in a powder form or nylon membranes derivatized with various functional groups (e.g. nitro group) (e.g. Schleicher and Schxc3xcll, Nytran; NEN, Gene Screen; Amersham Hybond M; Pall Biodyne).
The hybridized DNA or RNA is then detected by incubating the carrier, after thorough washing and saturation to prevent unspecific binding, with an antibody or antibody fragment. The antibody or antibody fragment is directed towards the substance incorporated into the nucleic acid probe during the derivatization. The antibody is in turn labelled. It is, however, also possible to use a directly labelled DNA After incubation with the antibodies, it is washed again in order to only detect specifically bound antibody conjugates. The determination is then carried out via the label of the antibody or antibody fragment according to well-known methods.
The detection of the LST-1 expression can be carried out for example as:
in situ hybridization with immobilized whole cells using immobilized tissue smears and isolated metaphase chromosomes,
colony hybridization (cells) and plaque hybridization (phages and viruses),
Northern hybridization (RNA detection),
serum analysis (e.g. cell type analysis of cells in serum by slot-blot analysis),
after amplification (e.g. PCR technique).
The invention therefore includes a method for the detection of nucleic acids which code for a LST-1 protein which is characterized in that the sample to be examined is incubated with a nucleic acid probe which is selected from the group comprising
a) the DNA sequences shown in SEQ ID NOS:1 and 2 or a complementary sequence to these,
b) nucleic acids which hybridize under stringent conditions with one of the sequences from a), the nucleic acid probe is incubated with the nucleic acid from the sample and the hybridization of the nucleic acid in the sample and nucleic acid probe is detected, if desired, via a further binding partner.
Thus, LST-1 is a valuable prognostic marker in tumor diagnostics (metastasis, progress) and an activity marker for cell proliferation, especially for T cell leukemic cells.
The human histiocytic lymphoma cell line U-937 was accorded the deposit number DSM ACC 5 and has since Apr. 3, 1990 been contained in, and freely accessible at, the public collection of Deutsche Samnlung von Mikroorganismen und Zeilkulturen GmbH, Mascheroder Weg 1b , D-38124 Braunschweig.
Plasmid pLST-1 which contains the LST-1 gene was deposited by Boehringer Mannheim GmbH at the Deutsche Sammlung von Mikroorganismen und Zeilkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig on May 24, 1995 and assigned the number DSM 10011.